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Ullah, S.;  Khalil, A.A.;  Shaukat, F.;  Song, Y. Biomedical Properties of Polysaccharides. Encyclopedia. Available online: https://encyclopedia.pub/entry/27473 (accessed on 27 July 2024).
Ullah S,  Khalil AA,  Shaukat F,  Song Y. Biomedical Properties of Polysaccharides. Encyclopedia. Available at: https://encyclopedia.pub/entry/27473. Accessed July 27, 2024.
Ullah, Samee, Anees Ahmed Khalil, Faryal Shaukat, Yuanda Song. "Biomedical Properties of Polysaccharides" Encyclopedia, https://encyclopedia.pub/entry/27473 (accessed July 27, 2024).
Ullah, S.,  Khalil, A.A.,  Shaukat, F., & Song, Y. (2022, September 22). Biomedical Properties of Polysaccharides. In Encyclopedia. https://encyclopedia.pub/entry/27473
Ullah, Samee, et al. "Biomedical Properties of Polysaccharides." Encyclopedia. Web. 22 September, 2022.
Biomedical Properties of Polysaccharides
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This entry is adapted from the peer-reviewed paper 10.3390/foods8080304

Polysaccharides are considered as vital bio-macromolecules for all living organisms, which are structurally comprised of homo or hetero monosaccharides and uronic acids connected with glycosidic linkages.

bioactive polysaccharides extraction biomedical applications

1. Introduction

Polysaccharides are considered as vital bio-macromolecules for all living organisms, which are structurally comprised of homo or hetero monosaccharides and uronic acids connected with glycosidic linkages [1][2][3]. They are predominantly found in various parts of plants, animals, fungi, bacteria, and seaweed, and play a critical role in numerous physiological functions of life [4]. Polysaccharides along with lipids, proteins, and polynucleotides are classified as the most pivotal four macromolecules in life sciences. Bioactive polysaccharides are known as polysaccharides produced from living organisms, and/or are functionalized from sugar-based materials possessing biological effects on organisms. Moreover, during the last decades, bioactive polysaccharides have been investigated as therapeutic agents against many chronic diseases due to their biodegradability, non-toxic nature, and biocompatibility [5]. Studies have shown that polysaccharides possess a wide range of pharmacological perspectives such as antioxidative, antitumor, antimicrobial, anti-obesity, hypolipidemic, antidiabetic, and hepato-protective properties [6][7][8]. They have been investigated extensively for the development of novel products in the field of cosmetics, food, medicine, petrochemicals, and paper [3][9][10]. Particularly, in the medicinal industry, polysaccharides are mostly used as pharmaceuticals and medical biomaterials (hypoglycemic, anti-osteoarthritic, and anticancer products) to curtail the effect of respective metabolic syndromes [9][11].
The potentiality of bioactive polysaccharides is strongly influenced depending upon their configuration and chemical structure. Nevertheless, the macromolecular configurations of plant cellular polysaccharides, particularly hetero polysaccharides (hemicelluloses), are very complex owing to the occurrence of various monosaccharides acting as isobaric stereoisomers [12][13]. Additionally, polysaccharides present in plants, microorganisms (bacteria, fungi, and yeasts), animals, and algae are chemically and/or physically bound with various other biomolecules like lignin, proteins, lipids, polynucleotides, and a few minerals [14]. Hence, to understand the importance of bioactive polysaccharides in the domain of life sciences resulted in the multi-disciplinary collaboration of scientists from the fields of microbiology, phytology, glycol-biology, nutrition, food sciences, and glycol-medicine [5]. Polysaccharides both in the simple and complex glycol-conjugated form are renowned for various bioactive functions, for instance, antivirus, antioxidant, antimicrobial, anticancer, antidiabetic, reno-protective, immunomodulatory, and anti-stress perspectives.

2. Sources of Bioactive Polysaccharides

Bioactive polysaccharides are categorized broadly depending upon their sources, structure, applications, solubility, and chemical composition. On the basis of chemical composition, they are characterized as homopolysaccharides (homoglycans) and heteropolysaccharides (heteroglycans). Homoglycans comprise of a single type of monosaccharide, such as glycogen and cellulose which consists of glucose molecules, whereas heteroglycans are made up of a different type of monosaccharides, for example, heparin and chondroitin sulfate (CS) [15].
According to the glycosides linked on to the glycan, polysaccharides can also be classified as proteoglycans and glycoproteins, glycolipids, and glycoconjugates [16][17]. According to grouping based on origins, bioactive polysaccharides are usually classified as those derived from animals (chondroitin sulfate, heparin, and hyaluronan), plants (pectin, inulin, Ginseng polysaccharides, xylans, and arabinans), bacteria (exopolysaccharides, capsular polysaccharides, and peptidoglycans), lichen, fungi, and algae.

3. Extraction and Quantification of Bioactive Polysaccharides

In general, bioactive potentials of naturally occurring polysaccharides are greatly dependent upon their molar mass and level and distribution of groups/side chains on the backbone. Therefore, isolation of polysaccharides from complex cellular plant matrices while keeping their bioactivity intact is of great significance. During the last decade, various innovative green extraction techniques (microwave-assisted, ultra-sonication, supercritical fluid extraction, and hot water extraction) are in practice for isolation of bioactive polysaccharides. These techniques have acquired great attention of scientists and researchers mainly due to their increased extraction rates, cost-effective nature, enviro-friendly characteristics, and structure preservative potentials [18][19][20].
Numerous scientific investigations have been implemented for the extraction and isolation of bioactive polysaccharides. The type of method adopted defines the physicochemical properties and antioxidant potential of isolated polysaccharides. For this purpose, a group of scientists compared the structural and antioxidant properties of bioactive polysaccharides extracted from Barrenwort (Epimedium acuminatum) through various extraction techniques (microwave-assisted, enzymatic, hot water extraction, and ultrasound-assisted extraction). They were of the view that bioactive polysaccharides isolated from hot water extraction had the highest antioxidant properties as compared to those extracted from other techniques, while their physicochemical properties were the same [21]. The hot water extraction method used in combination with various latest techniques (enzymatic pre-treatment, microwave, and ultrasound-assisted) are useful in increasing the yield and extraction productivity of polysaccharides. Likewise, enzymatic pre-treatment of raw material prior to extraction resulted in reduced extraction time, minimized the use of extraction solvents, preserved the bioactivities of the polysaccharides, and was energy efficient as compared to non-enzymatic pre-treated techniques [22]. In recent times, various ionic liquids have been formulated which aids in extraction of polysaccharides in a shorter time and at lower temperatures [23].
In addition, this purification of polysaccharides from the crude extract is really of great importance as the linkage among structure and safety of products formed for food, pharmaceutical, and biomedical application depends on this. Purification could be achieved by using various techniques (gel filtration, ion exchange and affinity chromatography, ethanolic precipitation, and fractional precipitation), individually or in combination [24].

4. Biomedical Applications

Polysaccharides and their derived compounds are medicinally more preferred as compared to synthetic polymers owing to their biodegradability, non-toxic nature, biocompatibility, and low processing expenses. Mentioned benefits related to polysaccharides isolated from natural sources make them a valuable ingredient in the fields of pharmaceuticals, nutraceuticals, food, and cosmetic industries. At the present time, polysaccharides are been used in healthcare and disease control, while various novel areas have also been discovered like in cancer diagnosis, inhibition, and treatment; in drug delivery; in anti-bacterial and anti-viral perspectives; and in tissue engineering [25][26].

4.1. Anti-Microbial and Antiviral

Various clinical investigations have authenticated that oral administration of pectin to infants and children significantly reduced diarrhea and other intestinal infections. This may be because of the decreased concentration of pathogenic bacteria like Citrobacter, Salmonella, Enterobacter, Shigella, Proteus, and Klebsiella [27]. A linear relationship has been documented among the concentration of probiotics and intestinal health [28].
The bioactive potential of fucoidans—a sulphated polysaccharide derived from marine brown seaweeds—have demonstrated noteworthy anti-viral potential against the cytomegalovirus, HIV, and HSV (herpes simplex virus) [29]. Additionally, few other seaweed-extracted polysaccharides like sulphated rhamnogalactans, carrageenans, and fucoidans have shown an inhibitory effect on viruses (HSV and HIV). Fucoidan comprises of a large quantity of L-fucose and sulphate groups along with fractions of galaturonic acid, xylose, mannose, and galactose. Undaria pinnatifida (marine brown alga) contains fucoidans and have been used in bone health supplementation mainly due to stimulation of osteoblastic cell differentiation. This sulphated polysaccharide has also been known to possess preventing action on UV-B-induced matrix metalloproteinase-1 (MMP-1) expression by inhibiting the ERK (extra-cellular signal regulated kinases) pathways. Therefore, it could be utilized as a functional ingredient in dermal ointments to prevent from skin photo-aging [30].
Some of the other fractions of algae have properties of virucidal and enzyme inhibitory activity inhibiting the formation of the syncytium. Besides, the sulfate group present is necessary for the anti-HIV activity and potency increases with the degree of sulfation.

4.2. Anti-Tumor/Cancer

Numerous scientists have explored dietary fibers as possessing potent anti-cancer properties. Amongst all, pectin has been investigated to reduce cancerous cell migration and tumor growth in a rat model that were administrated with modified citrus pectin [31]. This may be due to binding of pectin to galectin-3, which results in inhibitory action on some of its functional activities [32]. Anti-tumor mode of actions associated with dietary pectin are related to their immune-potentiation, probiotic properties, tumor growth inhibition, anti-mutagenic potential, and regulatory action of transformation-related oncogenes [33][34]. Anti-tumor mechanisms associated with pectin could be due to cellular immunological potential [35].
According to a study, ginseng polysaccharides were found to have a stimulating effect on DCs (dendritic cells) causing an elevated formation of IFN-g (interferon-g) [36]. It has also been documented that acidic ginseng polysaccharides (GPs) enhanced the production of cytotoxic cells against tumors and promoted macrophages for the production of Th1 and Th2 (helper type 1 and 2) cytokines [37][38]. Depending upon disease environment or timing of treatments, ginseng polysaccharides extracted from Panax ginseng demonstrated immuno-modulating perspectives mainly in an immunosuppressing or immuno-stimulating manner [39]. Acidic GP also revealed modulating action on the concentrations of antioxidative enzymes like GPx (glutathione peroxidase) and SOD (superoxide dismutase) probably due to induction of regulating cytokines [40][41]. Likewise, Lemmon et al. [41] found that the immuno-stimulating potential of acidic GPs isolated from American ginseng (Panax quinquefolius) was actually mediated by polysaccharides having molecular weight more than 100 kDa [42].
Furthermore, scientists have proven the fact that heparin administration may also have a beneficial impact on cancer and inflammation. Anti-cancerous, anti-inflammatory, and anti-tumor properties associated with heparin and its low molecular weight species are owing to the pathological functions of heparan sulfate (HS) chains of proteoglycan structure (HSPGs). Outcomes of an investigation validated that heparin transfers GRs (growth factors) stored by HS chains of HSPGs in the ECM (extracellular matrix) and on cell surfaces. Full-size heparin has potent pro-angiogenic properties as it increases the production of ternary complexes of heparin bound FGF2 and VEGF with GF receptors [43].

4.3. Anti-Obesity and Hypocholesterolemia

Numerous trials have shown a direct relationship between consumption of dietary fiber, rich diet/dietary fiber supplementation, and weight loss [44][45][46][47][48]. According to a meta-analysis comprising of 22 clinical trials, it was documented that a 12 g increase in the content of daily fiber intake resulted in a 10% decrease in energy intake along with a 1900 g decline in body weight [49]. More precisely, the administration of glucomannan (1.24 g/day) along with energy-restricted diet for five consecutive weeks caused a significant decrease in body weight as compared to the placebo group [50].
In a clinical trial on healthy volunteers, a drink containing oat β-glucan (10.5 g/400 g and 2.5 and 5 g/300 g) enhanced fullness sensation as compared to fiber-free drink [51][52]. Likewise, in healthy adolescents subjected to biscuits enriched with barley β-glucan (5.2%) helped in suppressing appetite ratings as compared to control biscuits [53]. Similarly, administration of bread formulated by barley β-glucan (3%) to volunteers resulted in decrease of hunger and increased satiety and fullness. This also resulted in a noteworthy decrease in energy intake at successive lunches [54]. On the other hand, a bar prepared from barley β-glucan (1.2 g) subjected to healthy volunteers did not change scores for energy intake and appetite scores as compared to control bars [55]. Effects of β-glucan on satiety depends upon the concentration, molecular weight (31–3100 kDa), solubility, and food carrying it [56].
Furthermore, a group of scientists investigated the hypocholesterolemic perspectives of a dietary supplement comprising equal content of konjac glucomannan (KGM) and chitosan [57]. The concentration of serum total cholesterol and low-density lipoprotein cholesterol (LDL-c) significantly reduced at the end of the trial (28th day). Fecal excretion of bile acids and neutral sterol were observed more at the commencement of the study as compared to the initiation of the study. Similarly, Chen et al. [58] investigated the impact of KGM supplementation (3.6 g/day) on levels of glucose and lipid biomarkers in hypercholesterolemic type-2 diabetic patients. Twenty-two diabetic patients having increased serum cholesterol content were selected for this study. As compared to the placebo group, KGM supplemented group showed decreased levels of LDL-c (20.7%), fasting glucose (23.2%), serum cholesterol (11.1%), and Apo-B (12.9%). Fecal bile acid and neutral sterol content were elevated significantly by 75.4% and 18.0%, respectively. Results of all the mentioned trials revealed that KGM supplementation could assist in the treatment of hypercholesterolemic diabetic patients [59].

4.4. Anti-Diabetic

Scientific evidences have shown that β-glucan can contribute to control glycemic responses. Numerous factors are found to affect such interactions like the nature of the food, concentration, and molecular weight of β-glucan. Among all these, the dose of β-glucan is considered to be the most important factor in regulating the impact of fiber on glycemic responses. As compared to other fibers, a small dose of β-glucan is sufficient to reduce the insulin and postprandial glucose responses in type 2 diabetic [60][61], healthy [62][63], and hyperlipidemic subjects [64]. Studies have revealed that consumption of breakfasts comprising of 4, 6, and 8.6 g of β-glucan momentously reduced the mean concentration of serum insulin and glucose as compared to control non-insulin-dependent diabetic mellitus subjects [60]. The content of exogenous glucose was noticed as 18% less in a polenta meal containing oat β-glucan (5 g) as compared to a control polenta meal without oat β-glucan-subjected individuals [65]. Likewise, consumption of a meal consisting of 13C-labelled glucose and β-glucan (8.9 g), for a period of three days, reduced (21%) the levels of exogenous 13C-glucose in plasma as compared to control meal having no β-glucan [56][66].

4.5. Gastro-Protective

An experimental trial conducted by means of two diverse types of resistant starches (one a high amylose granular resistant corn starch and the other was high amylose non-granular, dispersed, and retrograded resistant corn starch) to evaluate the influence on blood lipid concentration, fecal SCFA and bulking, and glycemic indexes. This study also comprised of supplements containing low fiber control and high fiber control. Outcomes of this trial revealed that high fiber control (wheat bran) and both resistance starches subjected groups showed an elevation in the fecal bulk as compared to the low fiber control group. Likewise, the average ratio of fecal SCFAs and butyrate had progressive effects on colon health. Xanthan gum may also be used in milk as a prebiotic for lactic acid bacteria. Similar trials regarding prebiotics have demonstrated protective implications on the sustainability of cultures under the presence of bile salts and refrigeration and low pH conditions. According to a study, guar gum has the capability to change lipoprotein and postprandial lipid compositions. Supplementation of guar gum has an influence on lipoprotein composition, lipemia, and postprandial glycaemia [67].

4.6. Immune Modulatory

Ginseng polysaccharides (GPs) have not only been known to possess immune-stimulating perspectives but also are found to suppress the proinflammatory responses. According to a recent study, novel neutral polysaccharide (PPQN) derived from an American ginseng root was documented to have a suppressing effect against inflammation. This activity was reported due to the inhibitory effect of isolated polysaccharide on inflammatory-related mediators such as cytokines (IL-1b, IL-6, TNF-a) and NO (nitric oxide) in comparison with LPS (lipopolysaccharide) treatment. Owing to this mode of action, novel neutral polysaccharide isolated from an American ginseng root could be used in modulating numerous inflammatory-related health implications (tumor, cancer, etc.) [68]. Similarly, another study reported the inhibitory influence of ginseng polysaccharides on immunological responses noticed in collagen-induced arthritic subject [69]. P. quinquefolius (American ginseng) is extensively used for the preparation of numerous herbal products. Extracts of P. quinquefolius were found to suppress the immune-inflammatory response, reduced the activity of neutrophils, induced the formation of cytokines in the spleen, and elevated the production of splenic-B lymphocytes and bone marrow [70][71][72][73].
Platycodon grandifloras is an herbaceous plant which is used as folk medicines since ancient times to curb various diseases like asthma, bronchitis, and pulmonary tuberculosis. Proximate composition of P. grandifloras reveals that it is a rich source of carbohydrates (90%), protein (2.4%), ash (1.5%), and fat (0.1%). Polysaccharides extracted from roots of P. grandifloras have been reported to possess antidiabetic, hypolipidemic and hypocholesterolemic properties [74]. Furthermore, the inulin-type polysaccharides isolated from P. grandifloras (PGs) roots validated the immune-modulating impact on macrophages and B-cells, but had no effect on T-cells [75].

4.7. Anti-Inflammatory

Astragalus polysaccharides (APS) are known to possess anti-inflammatory effects on cytokines of CD4+ Th (T-helper) cells. In in-vitro antidiabetic models, an Astragalus polysaccharide has potentiated the lowering effect on the expression of T-helper 1 (Th1) and regulated the imbalance of Th1 and Th2. APS has reported to significantly enhance the gene expression of peroxisome-proliferator-activated receptor gamma (PPAR-γ) in a concentration-time dependent manner [76] and stimulated superoxide dismutase (SOD) anti-oxidative mechanism in type-1 diabetes mellitus (DM) models [77][78]. Moreover, APS reduced the expression of iNOS (inducible nitric oxide synthase) [31]. These inflammatory markers (NO, PPAR-γ, SOD, and iNOS) amongst diverse roles also perform numerous functions in regulating and stimulating inflammatory response [79].
Water-soluble sulfated polysaccharides (WSSPs) isolated from marine algae are also classified as anti-inflammatory compounds. On the other hand, very few pieces of evidence are present regarding anti-inflammatory perspectives of seaweed based sulfated polysaccharides. In vitro and in vivo studies have revealed that Gracilaria verrucose- and Porphyra yezoensis-derived sulfated polysaccharides stimulated the respiratory burst and phagocytosis in experimented mouse macrophages [30]. Orally administrated chondroitin sulfate (CS) isolated from cartilage of Skate (Raja kenojei) affected arthritic conditions in a dose-dependent manner in chondroitin sulfate-treated groups. Pre- and post-treated groups that were subjected to CS (1000 mg kg−1) revealed momentously decreased clinical scores as compared to vehicle treated groups. CS administration decreased the infiltration of inflammatory cells and prohibited from paw and knee joint destruction. Moreover, the results of RT-PCR showed that CS ingestion significantly repressed the expression of IL-1b (interleukin-1b), IFN-c (interferon-c), and TNF-α as compared to vehicle administrated group. The CS-treated group reduced the formation of rheumatoid arthritis responses (IgG and IgM) in collagen-induced arthritic mice (CIA) model. Outcomes of this study authenticate the shielding potential of chondroitin sulfate in CIA mice mainly due to the inhibitory effect of pro-inflammatory cytokines formation [80].

4.8. Neuro-Protective

Acanthopanax senticosus derived polysaccharides comprised of uronic acid (22.5%), proteins (18.7%), and carbohydrates (58.3%). It could be established that Acanthopanax-based polysaccharides may not only help in improving symptoms regarding nervous defects but also reduced the infarct volume and water content of the brain in rats having cerebral ischemia–reperfusion injury. Additionally, polysaccharides isolated from A. senticosus elevated SOD, IL-10, and GSH-Px concentration and reduces the levels of TNF-α, IL-1, and MDA in brains tissues of experimented rats. Conclusively, bioactive polysaccharides extracted from A. senticosus protected brain damage due to antioxidative potential and inhibitory action on stimulation of inflammatory cytokines [81].

4.9. Anti-Oxidant

Bioactive acidic polysaccharides extracted from Polygonum multiflorum showed significant antioxidative properties (hydroxyl peroxide, superoxide anion radical, and hydroxyl radical), protein glycation and lipid oxidation. In addition to this, the intraperitoneal (i.p.) administration of P. multiflorium-based polysaccharides may increase the serum concentration of antioxidative characteristics in cyclophosphamide-induced anemic mice. Results of this study validate the use of P. multiflorium as a novel antioxidant tool to prevent oxidation [82]. Sulfated polysaccharides not only act as dietary fiber but also act as a natural antioxidant agent. They are responsible for the antioxidant properties possessed by marine algae. Various studies have recognized the use of numerous classes of SPs (alginic acid, Fucoidan, and laminaran) as potent antioxidative agents. Antioxidative potential of SPs has classified by multiple in-vitro methods such as DPPH, FRAP, NO, ABTS radical scavenging, superoxide radical scavenging assay, and the hydroxyl radical scavenging assay. Additionally, Xue et al. [83] stated that many marine-based sulfated polysaccharides have shown antioxidant potential in organic solvents and a phosphatidylcholine-liposomal suspension [30].

4.10. Tissue Engineering

Application of bioactive polysaccharides and their derivatives in the field of tissue engineering (cell differentiation, cell adhesion, cell remodeling, cell proliferation, and cell responsive degradation) has opened new horizons in medical research, and therefore impelled the researchers to regenerate new tissues and define the structure of cellular growth [25]. Various bioactive polysaccharides including starch, chitosan, chondroitin sulfate, alginate, cellulose, chitin, hyaluronic acid, and their derivatives are being used as biomaterials in applications for tissue engineering [84]. Application of these bioactive polysaccharides as scaffolds in tissue engineering are required to accomplish some requirements such as non-toxicity, biodegradability having controlled the rate of degradation, biocompatibility, structural integrity, and suitable porosity [25].
Chitosan and chitin have all the required potential to act as scaffolds for tissue engineering mainly due to their mechanical strength, degradability, and immunogenicity. Hence, for tissue engineering they are being developed as 3D-hydrogels, free standing films, porous sponges, and fibrous scaffolds, inside which for in-vitro/in-vivo cultures the most suitable cell types are needed [85]. Designing of 3D-chitin/chitosan-based hydrogels and sponge scaffolds, and 2D-scaffolds for the purpose of cartilage and tendon regenerations, for encapsulation of stem cells ensuring their therapeutic application, and for utilizing these as a tool for regenerative medicine have been reported in numerous researches [86][87]. Furthermore, for bone regeneration purpose, the tissue engineering industry has formulated combinations of chitosan and hydroxyapatite and grafted chitosan and carbon nanotubes [88]. Along with this, numerous other bioactive polysaccharides like cellulose, hyaluronic acid, and starch have also been studied in detail to validate their use as a biomaterial for skin, bone, and cartilage tissue engineering [89].

4.11. Wound Healing and Wound Dressing

Numerous bioactive polysaccharides (alginate, chitin, hyaluronan, chitosan, and cellulose) are used for the preparation of wound healing materials owing to their intrinsic bio-compatible, less toxic, and pharmaceutical activities [90][91]. For instance, hyaluronan is a vital extracellular component possessing distinctive viscoelastic, hygroscopic, and rheological characteristics are well known for its tissue repairing properties owing to their physicochemical potentials and specific interaction with cells and extracellular matrices. It is documented earlier that hyaluronan has a multidimensional role regarding the repairing process of cell or wound healing specifically inflammation, granulation, formation of tissues, re-epithelialization, and remodeling. Various hyaluronan-derived products like esterified, cross-linked, or chemically modified products are medicinally used for wound healing and tissue repairing purposes [92]. While designing bioactive material for tissue engineering their wound healing properties is of great interest.

4.12. Drug Delivery and Controlled Release

Application of bioactive polysaccharides as a novel agent in drug delivery and controlled release has also been studied by scientists owing to their least toxicity, minimum immunogenicity, and biocompatibility. Various naturally occurring polysaccharide-based drug delivery systems are in practice due to their targeted delivery/controlled release, shielding effect against premature degradation of drugs, improvement of intracellular transportation, enhancement of bioavailability of drugs, as well as delivery of small interfering RNA, antigens, and genes [93]. Delivery systems mentioned here usually possess covalent/ionic cross-linkages, poly-electrolyte complexes, conjugates of polysaccharides and drugs, and self-assembly [93]. Release of 3-D cross-linked drugs could be triggered by varying redox potential, pH, light, ions, temperature, and application of magnetic and/or electric fields [94]. Mainly the three most abundantly used polysaccharides i.e., alginate, chitin, cellulose, and chitosan are overviewed in detail as under in this portion.

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Samee Ullah
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Faryal Shaukat
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Yuanda Song
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